CN114438245B - SNP molecular marker linked with peanut bacterial wilt-resistant major QTL locus and application thereof - Google Patents
SNP molecular marker linked with peanut bacterial wilt-resistant major QTL locus and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular markers, in particular to an SNP molecular marker linked with a peanut bacterial wilt resistance major QTL locus and application thereof. The SNP polymorphism site in the SNP molecular marker BWRB03-R provided by the invention is located at 201 th site of the sequence shown as SEQ ID NO.1, and the polymorphism is G/A. Specifically, in the SNP molecular marker BWRB03-R, the genotype of the polymorphic site is A, the corresponding phenotype of the peanut is stronger bacterial wilt resistance, the genotype of the polymorphic site is G, and the corresponding phenotype of the peanut is weaker bacterial wilt resistance. By adopting the SNP molecular marker provided by the invention, the peanut material with bacterial wilt resistance can be selected in an auxiliary way, the breeding of the peanut variety with bacterial wilt resistance is promoted, and the breeding efficiency is improved.
Description
Technical Field
The invention relates to the technical field of molecular markers, in particular to an SNP molecular marker linked with a peanut bacterial wilt resistance major QTL qBWRB03 and application thereof.
Background
Peanut (Arachis hypogea L.) is one of the important oil crops. Bacterial wilt caused by ralstonia solanacearum is the most important bacterial disease that impairs peanut production. The ralstonia solanacearum invades the roots of the plants from the soil, expands upwards along the vascular bundle and breeds in large quantities, and finally, the plant ducts lose water conveying capacity and die quickly. In the production of peanuts, the disease death rate of plants in a general diseased region is 10-30%, the disease death rate of plants in a seriously diseased region can reach more than 80%, and in extreme cases, all plants die to cause failure. Therefore, effective prevention and control of bacterial wilt is one of the great demands for development of the peanut industry.
The breeding and the utilization of disease-resistant varieties are one of the most economic and effective measures for preventing and treating peanut bacterial wilt. The resistance phenotype of the peanuts to the bacterial wilt disease is quantitative resistance, and the discovery and utilization of the stable main effect QTL are the key basis for breeding bacterial wilt resistant varieties. By constructing a recombinant inbred line group, identifying bacterial wilt resistance in multiple environments, and carrying out QTL analysis by combining molecular marker genotypes, a stably expressed main effect QTL can be identified; the molecular marker linked with the QTL can be used for carrying out molecular marker-assisted selection, so that the breeding efficiency is improved.
At present, research on the QTL positioning of peanut bacterial wilt resistance is less, and only stable main effect QTL and molecular markers thereof are discovered on B02 chromosome. In order to further improve the resistance level of bacterial wilt, the discovery and utilization of a new disease-resistant QTL major site are of great importance.
Disclosure of Invention
The invention aims to provide a method for detecting a peanut bacterial wilt-resistant major QTL locus qBWRB03, which can be used for quickly judging the bacterial wilt resistance of peanuts.
Specifically, the invention provides a peanut bacterial wilt resistance SNP molecular marker BWRB03-R in a first aspect. More specifically, the SNP molecular marker BWRB03-R provided by the invention is linked with a peanut bacterial wilt-resistant major QTL locus qBWRB03.
The SNP molecular marker BWRB03-R provided by the invention is tightly linked with the bacterial wilt resistance major QTL locus qBWRB03 positioned on the B03 chromosome.
The SNP molecular marker BWRB03-R provided by the invention has polymorphic site G/A at 201 th site of the sequence shown in SEQ ID NO. 1.
In the SNP molecular marker BWRB03-R, the genotype of the 201 st polymorphic site is A, the genotype corresponds to the QBWRB03 site containing the bacterial wilt resistance major QTL, the genotype is G, and the genotype corresponds to the absence of the qBWRB03.
The SNP molecular marker BWRB03-R provided by the invention is obtained by amplification by using a primer pair shown in SEQ ID NO. 2-3.
In a second aspect, the invention provides a primer pair, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3. The primer pair provided by the invention is used for amplifying the SNP molecular marker BWRB03-R.
BWRB03-R primer set:
a forward primer sequence 5'-ATGAAAAGTGAAAAATGAAAAGTGAAAAG GGA-3' (shown as SEQ ID NO. 2),
the reverse primer sequence 5'-CAGCGGGGGCAAGTTCAC-3' (as shown in SEQ ID N O.3).
In a third aspect, the invention claims a reagent or a kit containing the primer pair.
According to the understanding of the technical personnel, the invention also claims the application of the SNP molecular marker BWRB03-R, or the primer pair shown in SEQ ID NO.2-3, or the reagent or the kit containing the primer pair shown in SEQ ID NO.2-3 in the detection of the peanut bacterial wilt resistance major QTL qBWRB03.
Specifically, in a fourth aspect, the invention provides a method for detecting a peanut bacterial wilt resistance major QTL qBWRB03, which comprises the following steps:
(1) Carrying out PCR amplification on the DNA of the peanut to be detected by utilizing a primer pair shown in SEQ ID NO. 2-3;
(2) Analyzing the genotype of the SNP molecular marker BWRB03-R in the PCR amplification product, and judging whether the peanut to be detected contains the bacterial wilt resistance major QTL qBWRB03 or not according to the genotype.
In the method for detecting the bacterial wilt resistance major QTL qBWRB03 of the peanut, if the genotype of the SNP molecular marker BWRB03-R is A, the peanut to be detected contains the bacterial wilt resistance major QTL qBWRB03; if the genotype of the SNP molecular marker BWRB03-R is G, the peanut to be detected does not contain qBWRB03.
The invention also claims the application of the SNP molecular marker BWRB03-R, or a primer pair shown in SEQ ID NO.2-3, or a reagent or a kit containing the primer pair shown in SEQ ID NO.2-3 in any one of the following applications:
(1) The application in peanut germplasm resource identification, improvement or molecular marker-assisted breeding;
(2) The application in early prediction of peanut bacterial wilt resistance;
(3) Application in screening of peanut with bacterial wilt resistance.
In a fifth aspect, the present invention provides a method for identifying resistance to peanut bacterial wilt, comprising:
(1) Carrying out PCR amplification on the DNA of the peanut to be identified by using a primer shown in SEQ ID NO. 2-3;
(2) Analyzing the genotype of the SNP molecular marker BWRB03-R in the PCR amplification product, and judging the bacterial wilt resistance of the peanut to be identified according to the genotype.
In the method for identifying the peanut bacterial wilt resistance, if the genotype of the SNP molecular marker BWRB03-R is A, the peanut to be identified has strong bacterial wilt resistance; the genotype of the SNP molecular marker BWRB03-R is G, and the bacterial wilt resistance of the peanut to be identified is weak.
The invention has the beneficial effects that:
the SNP molecular marker BWRB03-R linked with the peanut bacterial wilt resistant major effective site qBWRB03 is obtained by screening, a primer pair for detecting the SNP molecular marker is designed, and the method for detecting the SNP molecular marker of the peanut bacterial wilt resistant major effective site qBWRB03 is adopted, so that the peanut material resistant to bacterial wilt can be selected in an auxiliary manner, the breeding of peanut varieties resistant to bacterial wilt can be promoted, and the breeding efficiency can be improved.
The SNP molecular marker of the invention mainly detects whether the locus qBWRB03 resisting bacterial wilt exists, and the peanut material containing the locus qBWRB03 has stronger capacity of resisting bacterial wilt. In breeding application, ICG12625 or other material with qBWRB03 can be used as parent to perform hybridization, so that many individuals with or without qBWRB03 can be generated in offspring, individual material with qBWRB03 sites is selected by mark assistance, and then the individual material is selected with high yield and excellent quality, and finally a new peanut variety with bacterial wilt resistance can be formed.
Drawings
FIG. 1 is a map of the peanut bacterial wilt resistance major locus qBWRB03 in chromosome B03 of the present invention, wherein the vertical line represents peanut chromosome B03, and the horizontal short line segment on the right side of B03 represents the genetic recombination bin on the chromosome; left lateral short line segment represents the genetic distance between bins (cM); the major bacterial wilt resistance site qBWRB03 is positioned in the 1cM region between c13B013 and c13B014 of bin sites, and the right side of B03 lists the confidence interval of the QTL detected in 2019-2020 two years.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
This example provides the development process of SNP molecular marker BWRB03-R linked with peanut bacterial wilt resistance major QTL locus qBWRB03, as follows.
Test materials: the method comprises the steps of hybridizing a peanut bacterial wilt resistant variety flower 10 serving as a female parent and a peanut bacterial wilt resistant germplasm ICG12625 serving as a male parent, and obtaining a Recombinant Inbred Line (RIL) group containing 140 strains by a single seed transmission method.
And (3) phenotype identification: 140 strains of Zhonghua 10, ICG12625 and RIL groups were identified for bacterial wilt resistance in the Honan bacterial wilt disease nursery at the oil crop institute of Chinese academy of agricultural sciences in 2019 and 2020. A fully randomized block experimental design was used, 3 replicates. Each time, 20 plants were planted in 1 row per material, with a row spacing of 30 cm and a plant spacing of 10 cm. And adopting a standard field management mode. After all seedlings emerged, the total number of plants per material was counted. And after the bacterial wilt begins to develop diseases, counting the number of plants died of the diseases until the plants are harvested. And then calculating the survival rate: (total number of plants-number of dead plants)/total number of plants × 100%, the higher the survival rate, the better the resistance of the material to bacterial wilt.
Construction of genetic linkage map: extracting DNA of 140 single plant leaves of parent and RIL groups by a CTAB method; a300-500 bp Illumina Paired-end (PE) library was constructed for each sample DNA and sequenced by PE 150. The original data of the machine is subjected to quality control to obtain high-quality cl eandata, then BWA software is used for comparing the clean data to a tetraploid cultivation peanut reference genome (https:// www.peanutbase.org/data/public/Arachis _ hypogaea/Tifr unner. Gnm1.KYV3 /), and finally a HaplotpypeCaller module of GATK is used for carrying out SNP detection. Combining SNP sites with the same genotype into bins, and finally calculating the genetic map distance of the bins to obtain a final linkage map. The linkage map contained 2701 bin sites, with a total length of 1469.56 cM and an average inter-marker distance of 0.54cM.
Positioning of QTL: the Q TL analysis of survival rate is carried out by utilizing a region mapping method of WinQTLCart software, the survival rate phenotype data of two environments detect a main QTL locus qBWRB03 in a c13B 013-c 13B014 region on a chromosome B03, the LOD values of the main QTL locus qBWRB03 in the two environments are 4.82 and 5.11 respectively, and the phenotype interpretation rates are 12.01 percent and 10.5 percent respectively.
The confidence intervals of the two environments overlap each other and are located in an interval of 1cM (6.7-7.7 cM), and the position of the two environments is schematically shown in FIG. 1. Therefore, the peanut bacterial wilt-resistant major site qBWRB03 can be stably expressed in two-year experiments, and the resistance allele of the peanut bacterial wilt-resistant major site qBWRB03 is derived from ICG12625.
An SNP molecular marker BWR B03-R (SEQ ID NO. 1) is developed by utilizing a SNP (located in the reference genome B03 chromosome 125,198,196bp (G/A variation)) in a c13B 013-c 13B014 interval, and the second generation high-throughput sequencing of an amplification product detects the base composition of the SNP site: detecting A base in ICG12625, belonging to gene containing qBWRB03 bacterial wilt resistance allele; and G bases detected in the middle flower No. 10 do not contain qBWRB03 bacterial wilt resistance allele.
The primer designed for the molecular marker BWRB03-R provided in example 1 is shown in SEQ ID NO. 2-3.
Example 2
This example provides the detection of ICG12625 and 131 RILs of Zhonghua No. 10, which obtained survival data from both environments in example 1, using the SNP molecular marker BWRB03-R linked to the bacterial wilt resistance major site qBWRB03. The method comprises the following specific steps.
Using genome DNA as a template and a sequence shown in SEQ ID NO.2-3 as a primer, and amplifying an SNP molecular marker BWRB03-R by using a KAPA2G Fast Multiplex Mix kit. The PCR conditions were: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 15s, renaturation at 55 ℃ for 30s and extension at 72 ℃ for 30s for 30 cycles; finally, extension is carried out for 5min at 72 ℃, and heat preservation is carried out at 4 ℃. Adding a sequencing linker into the amplified product, then carrying out Paired-end 150bp (PE 150) sequencing by using an Illumina HiSeq platform, comparing a sequencing sequence to a reference sequence, and detecting the base of the SNP site. If the base of the SNP site detected by the RIL and the amplified fragment of ICG12625 are consistent, the strain contains the bacterial wilt resistance allele of qBWRB03. And simultaneously, the actually measured survival rate (the test method is shown as example 1) result and the molecular marker detection result are used for verification.
The results show that the SNP molecular marker genotypes of 62 RILs in 131 RILs are the same as ICG12625 (genotype classification 1,A), indicating that the SNP molecular marker loci qBWRB03 contain homozygous major bacterial wilt resistance QTL sites qBWRB03; the SNP molecular marker genotypes of 69 RILs were the same as those of Zhonghua No. 10 (genotype classification 2,G), indicating that no qBWRB03 was contained.
The T-test found that 62 rls containing qBWRB03 were significantly higher in survival identified in both 2019 and 2020 than 69 rls containing no qBWRB03 (table 1). Therefore, the SNP molecular marker BWRB03-R linked with the bacterial wilt resistance major locus qBWRB03 is used for predicting the peanut resistance to bacterial wilt.
TABLE 1 survival Difference in the selection of RIL strains Using the SNP molecular markers of the invention
* Indicates that at the 0.01 level there was a significant difference between genotype classification 1 and genotype classification 2. Genotype classification 1 was the same as ICG12625 and genotype classification 2 was the same as middle flower No. 10.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> institute of oil crop of academy of agricultural sciences of China
<120> SNP molecular marker linked with peanut bacterial wilt resistance major QTL locus and application thereof
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Claims (4)
1. The primer pair for amplifying the peanut bacterial wilt-resistant SNP molecular marker BWRB03-R is characterized in that the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3; the SNP molecular marker BWRB03-R is a polymorphic site G/A at the 201 st site of a sequence shown in SEQ ID NO. 1.
2. A reagent or a kit comprising the primer set according to claim 1.
3. The primer pair of claim 1, or the reagent or kit of claim 2, for use in any one of:
(1) The application in early prediction of peanut bacterial wilt resistance;
(2) Application in screening of peanut with bacterial wilt resistance.
4. The method for identifying the resistance of peanut bacterial wilt is characterized by comprising the following steps:
(1) Carrying out PCR amplification on the DNA of the peanut to be identified by using a primer pair shown in SEQ ID NO. 2-3;
(2) Analyzing the genotype of the SNP molecular marker BWRB03-R in the PCR amplification product, and judging the bacterial wilt resistance of the peanut to be identified according to the genotype;
(3) If the genotype of the SNP molecular marker BWRB03-R is A, the bacterial wilt resistance of the peanut to be identified is strong; the genotype of the SNP molecular marker BWRB03-R is G, and the bacterial wilt resistance of the peanut to be identified is weak.
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| CN113564161A (en) * | 2021-08-27 | 2021-10-29 | 河南省农业科学院 | Molecular marker closely linked with bacterial wilt resistance of cultivated peanuts and application |
| CN113736910A (en) * | 2021-10-12 | 2021-12-03 | 山东省花生研究所 | Linkage molecular marker of peanut single plant pod number main effect QTL site qPN7 and application thereof |
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| CN113564161A (en) * | 2021-08-27 | 2021-10-29 | 河南省农业科学院 | Molecular marker closely linked with bacterial wilt resistance of cultivated peanuts and application |
| CN113736910A (en) * | 2021-10-12 | 2021-12-03 | 山东省花生研究所 | Linkage molecular marker of peanut single plant pod number main effect QTL site qPN7 and application thereof |
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| Title |
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| 抗青枯病兼大果和高出仁率的花生新种质创制;李威涛等;《作物学报》;20200115;第46卷(第4期);第484-490页 * |
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